The continuous measurement of substances in biological fluids is of interest in the study and control of metabolic disorders. Electrode systems have been developed for this purpose whereby an enzyme-catalyzed reaction is monitored by an electrochemical sensor. In such electrode systems, the electrochemical sensor comprises an electrode with potentiometric or amperometric function in close contact with a thin layer containing an enzyme in dissolved or insoluble form. The thin layer may also include a co-enzyme.
In conventional practice, a semipermeable membrane separates the thin layer of the electrode containing the enzyme from the sample of biological fluid that includes the substance to be measured. The electrochemical sensor measures the concentration of the substance involved in the enzyme reaction. For example, the concentration of a co-enzyme or a reaction product can be determined. This concentration may be related to the substrate concentration in the sample by its stoichiometric relationship and by calibration of the electrode system.
A number of enzyme electrodes have been developed, and the operation of those electrodes varies depending on the nature of the enzyme reaction and the particular substance being measured. For example, enzyme electrodes include those that measure: (1) a reactant or product of the enzyme reaction; (2) the consumption of a co-enzyme based on the decrease of its initial concentration and (3) the amount of the reduced or oxidized form of a co-enzyme produced during the enzyme reaction.
The operation of a particular enzyme electrode depends on a number of parameters including diffusion processes, kinetics of the enzyme reaction and the type of electrochemical sensor. In particular, the operation of the electrode can be affected by the diffusion of substances through the semipermeable membrane.
Electrode systems that include enzymes have been used to convert amperometrically inactive substances into reaction products which are amperometrically active. Specifically, in the analysis of blood for glucose content, glucose (which is relatively inactive amperometrically) may be catalytically converted by the enzyme glucose oxidase in the presence of oxygen and water to gluconic acid and hydrogen peroxide. Hydrogen peroxide is anodically active and produces a current which is proportional to the concentration of hydrogen peroxide in the blood sample and thus to the concentration of glucose in the sample.
In a sample of undiluted whole blood, however, a molar excess of plasma glucose is present relative to the amount of plasma oxygen. As a result, if a semipermeable membrane is not included over the enzyme, the concentration of glucose in the sample relative to the concentration of oxygen will be so high that the glucose oxidase-catalyzed reaction of glucose and oxygen to gluconic acid and hydrogen peroxide will be oxygen limited.
The effect of an oxygen limited reaction is that the range of glucose concentrations that can be measured with such an electrode is very limited. In particular, linearity is not achieved above minimal concentrations of glucose. In a clinical setting, linear glucose levels must be obtained at glucose concentrations of at least up to about 500 milligrams per deciliter (mg/dl). Without a semipermeable membrane over the enzyme, linear glucose levels can be obtained only up to about 40 mg/dl. Thus, the purpose of the membrane over the enzyme in a glucose sensing electrode system is to limit the amount of glucose that passes or diffuses through the membrane. This extends the upper limit of linearity of glucose measurement from a low value without the membrane to a high value with the membrane.
The two fundamental diffusion processes by which a semipermeable membrane can limit the amount of a substance that passes therethrough are diffusion through the semipermeable membrane as a monolithic, homogeneous structure and diffusion through the semipermeable membrane as a porous structure. The processes of diffusion of substances through these different types of membranes differ considerably.
A semipermeable membrane can comprise a porous structure consisting of a relatively impermeable matrix that includes a plurality of "microholes" or pores of molecular dimensions. Transfer through these membranes is primarily due to passage of substances through the pores. In other words, the membrane acts as a microporous barrier or sieve.
Examples of materials that may be used to form such membranes include polyethylene, polyvinyl chloride, tetrafluoroethylene, polypropylene, cellophane, polyacrylamide, cellulose acetate, polymethyl methacrylate, silicone polymers, polycarbonate, cuprophane and collagen.
Selectivity in such a membrane can be explained on the basis of the molecular size of the diffusing substances. For substances much smaller than the diameter of the pores, passage of the substance through the membrane is relatively unimpeded. As the effective molecular diameter of the substance approaches the diameter of the pore, the pore will exert a drag on the diffusing substance, reducing its permeability to a value lower than that expected on the basis of the membrane porosity. If the molecules of the substance are too large, they will not pass through the membrane at all.
Since transfer is due primarily to passage of the substance through pores, the permeability is directly related to the size of the pores and to the molecular volume of the diffusing substance. As a result, there is little selectivity in the separation of two chemically or structurally related molecules, except when their molecular size is approximately the same as the size of the pore. When this occurs, there is the possibility that forces acting between the substance and the surface of the pore channel may influence the rate of transfer.
Also, the upper size limit to diffusion will be determined by the largest pore diameter, and the overall diffusion rate will depend on the total number of pores for movement of the substance.
Passage of a substance through a monolithic, homogeneous membrane, on the other hand, depends upon dissolution and diffusion of the substance as a solute through a solid, non-porous film. As used herein, the term "monolithic" means substantially non-porous and having a generally unbroken surface. The term "homogeneous", with reference to a membrane, means having substantially uniform characteristics from one side of the membrane to the other. However, a membrane may have heterogeneous structural domains, for example, created by using block copolymers, and still be characterized functionally as homogeneous with respect to its dependence upon dissolution rather than sieving to effect separation of substances. A monolithic membrane can thus be used to separate components of a solution on the basis of properties other than the size, shape and density of the diffusing substances. The membrane acts as a barrier because of the preferential diffusion therethrough of some substance (a solute).
Despite advances in membrane technology, devices that include semipermeable membranes which have been used to detect and measure the presence of a substance in a biological fluid have generally been restricted to laboratory environments. This is because the devices are generally large and complex and require extensive training to operate. In addition, these devices have been somewhat limited because of the difficulty in replacing a membrane used with the electrode.
A need exists for an improved device that selectively measures the presence and the amounts of particular substances in biological fluids. Such a device should accurately measure the amount of a substance in a sample without dilution or pretreatment of the sample. In addition, a basis for selecting appropriate membrane materials for use in such devices is needed. The device should also be easy to use and provide a means for replacing the membrane as necessary.